Critical point drying systems and methods for in situ tissue preservation

ABSTRACT

Methods and systems for preserving tissues in situ using critical point drying are disclosed. Such methods and systems are particularly applicable to the preservation of a deceased body, such as a deceased person or animal, with or without removal of internal tissues or organs. A fixative can be perfused through the vascular system of the body while blood is removed from the body. The exterior of the body can also be immersed in a bath of fixative. The fixative in the vascular system and the bath can be replaced by subsequent washes of buffer, de-ionized water, and/or alcohol. The alcohol-infused and fixated body can be disposed in a pressure chamber and subjected to a critical point drying process using carbon dioxide. After the critical point drying process, the body is in a preserved state.

FIELD

The present disclosure relates generally to the preservation of tissues,and, more particularly, to the preservation of tissues in situ usingcritical point drying.

SUMMARY

In general, perfusion of fixative together with critical point dryingcan be used to preserve a body, such as a deceased human or animal,without removal of internal tissues or organs. Fixative can be perfusedthrough the vascular system of the body through the heart while blood isremoved from the body. The exterior of the body can also be immersed ina bath of fixative. The fixative can be replaced by subsequent washes ofbuffer, de-ionized water, and/or alcohol. The body can be infused withand immersed in, for example, liquid carbon dioxide. Critical pointdrying of the body can then be performed by heating to a temperature andpressure at or in excess of the critical point (i.e., the criticaltemperature and pressure) of carbon dioxide. The carbon dioxide can thenbe exhausted, after which the body remains in a substantially preservedstate. In one or more alternative embodiments, critical point dryingalone (i.e., without any fixation) may be used to preserve certainbodies and/or tissues.

In one or more embodiments, a method for preserving a deceased body oran organ thereof can include perfusing the body or the organ withfixative delivered via the vascular system thereof. After the perfusingwith fixative, the body or the organ can be perfused with liquid carbondioxide delivery via the vascular system thereof. After the perfusingwith liquid carbon dioxide, the body or the organ can be heated in asealed chamber until the temperature and pressure in the chamber meetsor exceeds the critical point for carbon dioxide.

In one or more embodiments, a system for preservation of a body or anorgan thereof can include a pressure chamber having at least one fluidinlet and a vascular inlet line. The vascular inlet line can connect tothe vascular system of the body or the organ. The pressure chamber canbe sized and shaped so as to allow the body or the organ to be enclosedtherein. A flow module can supply at least a transitional fluid to thepressure chamber through the at least one fluid inlet and to thevascular system through the vascular inlet line. For example, thetransitional fluid can be carbon dioxide. A temperature module cancontrol the temperature of the pressure chamber. A system controller cancontrol the flow module to fill the pressure chamber and the vascularsystem with transitional fluid. The system controller can additionallycontrol the temperature module to heat the transitional fluid above itscritical point temperature and to pressurize the transitional fluidabove its critical point pressure. The pressure chamber can beconstructed to withstand at least the critical point temperature andpressure of the transitional fluid.

In one or more embodiments, a method for preserving a body or an organthereof can include perfusing the body or the organ with liquid carbondioxide by way of the vascular system while the body or the organ is ina scaled pressure chamber. The external surfaces of the body or theorgan can be exposed to liquid carbon dioxide while the body is in thesealed pressure chamber. The body or the organ can be heated in thesealed pressure chamber until the temperature and pressure in thepressure chamber meets or exceeds the critical point for carbon dioxide.The method can further include, after the heating, exhausting carbondioxide from the pressure chamber and the body or the organ therein.

In one or more embodiments, a method for preserving a recently deceasedhuman body can include connecting a perfusion inlet and outlet to thevascular system of the deceased human body. The method can furtherinclude flowing a fixative into the vascular system of the deceasedhuman body and immersing the deceased human body in fixative. Thefixative can include, for example, glutaraldehyde or formaldehyde. Themethod can also include flowing a buffer solution into the vascularsystem and immersing the deceased human body in buffer solution. Inaddition, the method can include flowing water into the vascular systemand immersing the deceased human body in water. The method can includeflowing alcohol into the vascular system and immersing the deceasedhuman body in alcohol. The method can additionally include flowingliquid carbon dioxide into the vascular system and immersing thedeceased human body in liquid carbon dioxide in a sealed pressurechamber. Further, the method can include heating the deceased human bodyin the sealed pressure chamber until the temperature and pressure in thepressure chamber meets or exceeds the critical temperature and criticalpressure for carbon dioxide.

Objects and advantages of the present disclosure will become apparentfrom the following detailed description when considered in conjunctionwith the accompanying drawings.

BRIEF DESCRIPTION OF DRAWINGS

Embodiments will hereinafter be described in detail below with referenceto the accompanying drawings, wherein like reference numerals representlike elements. The accompanying drawings have not necessarily been drawnto scale. Where applicable, some features may not be illustrated toassist in the description of underlying features.

FIG. 1 is a schematic diagram showing generalized features of a methodfor in situ tissue preservation according to one or more embodiments ofthe disclosed subject matter.

FIGS. 2A-2B is a process flow diagram of a method for in situ tissuepreservation according to one or more embodiments of the disclosedsubject matter.

FIG. 3 is a schematic diagram showing an arrangement for perfusion of abody according to one or more embodiments of the disclosed subjectmatter.

FIG. 4 is a schematic diagram of a system for perfusion of a bodyaccording to one or more embodiments of the disclosed subject matter.

FIG. 5A is a plan view of a chamber for preserving a body according toone or more embodiments of the disclosed subject matter.

FIG. 5B is a cross-sectional view of the chamber of FIG. 5A along lineB-B.

FIG. 6 is a schematic diagram of a system for in situ tissuepreservation according to one or more embodiments of the disclosedsubject matter.

FIGS. 7A-7C show examples of various configurations for standalonepreservation systems, according to one or more embodiments of thedisclosed subject matter.

FIG. 8 is a process flow diagram of an alternative method for in situtissue preservation according to one or more embodiments of thedisclosed subject matter

DETAILED DESCRIPTION

To arrest the decay and decomposition of a recently deceased body, thecells of the body can be exposed to a fixative. The exterior of the bodyis immersed or submerged in the fixative. Cells internal to the body canbe exposed to the fixative by perfusing the fixative through thevascular system. In other words, fixative may be flowed through thecirculatory system. Access to the vascular system may be achieved by anappropriate incision to the thoracic cavity and coupling perfusiontubing to the heart. Perfusion of fluids into the deceased body may alsobe accomplished via an arterial connection or other access point, suchas, but not limited to a fistula, a graft, or a catheter.

Fixative internal and external to the body can be sequentially replacedby subsequent washes of buffer, water, and/or alcohol. With the body ina pressure chamber, the alcohol can be replaced with a critical pointdrying agent, such as but not limited to carbon dioxide. Critical pointdrying of the body can be performed by heating the pressure chamber andthe body therein past the critical point (i.e., the critical temperatureand pressure) of the drying agent and then exhausting the drying agentfrom both the body and the chamber. In alternative embodiments, criticalpoint drying alone may be used to preserve certain bodies or tissues,such as, but not limited to, vegetation and non-vascular organisms.

According to embodiments of the systems, methods, and devices disclosedherein, a deceased body may be preserved indefinitely. The cellularstructure of the body can maintained, thereby allowing diagnostic oranatomical analysis, such as for medical training or research. Becausethe decomposition has been arrested, the deceased body may be maintainedindefinitely in the same condition as it was immediately post-mortem. Inaddition, cellular material and structure are preserved by the fixationprocess. Future technological advances may thus be able reverse thefixation process and reanimate the deceased body.

In embodiments, the systems, methods, and devices described herein canbe applied to the long-term preservation of recently deceased humans, asan alternative to conventional embalming and/or cryonics processes.Although certain examples are explicitly discussed herein with respectto deceased humans, the methods, systems, and devices are equallyapplicable to the preservation of a wide range of biological organismsand structures, including, but not limited to, deceased animals,insects, and plants, as well as tissues, organs, and portions thereof.

Referring now to FIG. 1, a generalized method 100 for in situ tissuepreservation is shown. The method 100 can begin with an optionalpreparation step 102. In the optional preparation step 102, the body maybe cleansed in preparation for fixation, such as by washing and/ordisinfecting the exterior of the body. Certain interior cavities of thebody may also be cleansed. For example, an enema or colonic may beperformed to remove fecal matter, bacteria, gut flora, and/or otherforeign organisms from the lower gastrointestinal tract. Undigested orpartially digested food may also be removed from the uppergastrointestinal tract. However, such a step 102 is not required and maybe omitted.

In addition, the preparation may include repairing damage to thevascular system. In some circumstances, the deceased body may haveundergone damage due to, for example, an accident, trauma, autopsy,injury, or other occur occurrence. In such circumstances, the damage canbe surgically repaired to allow perfusion through the vascular system.Alternatively, the damage can be sealed or bypassed with appropriatetubing. In still another alternative, the body may be perfused usingmulti-point injections, for example, using separating injections pointin the iliac or femoral arteries, the subclavian or axillary vessels,and/or the common carotids. The preparation at 102 can include makingthe necessary fluid connections to these injection sites in preparationfor later perfusion.

At 104, the body can undergo fixation (e.g., via perfusion) byintroducing a fixative into the body and over the external surfaces ofthe body. Various fixatives can be used to achieve the fixation. Forexample, the fixative can be 1.5% glutaraldehyde in a phosphate buffer.In another example, the fixative can include a combination of fixatives,such as 1.5% glutaraldehyde and 1% formaldehyde in a buffer solution ata pH of approximately 7.3. For certain applications, impurities in thefixative may adversely affect cell structure or preservation properties.Thus, the fixative may be purified prior to use. While the fixation step104 may enhance the preservation effect in some application, it can beomitted in other applications. For example, fixation may not benecessary when preserving certain organisms, such as vegetation.

At 106, the fixative can be flushed by one or more washes. Thus, thefixative and remnants thereof can be removed from the body inpreparation for subsequent critical point drying. The flushing step 106can be achieved using multiple washes of buffer and/or water.

For example, a buffer can be perfused through and flowed over the bodyfor a sufficient volume to allow complete removal of the fixative, afterwhich water can then be perfused through and flowed over the body for asufficient volume to allow complete removal of the buffer.Alternatively, a sufficient volume of buffer can be introduced tocompletely fill the body's vascular system and to completely cover thebody. After a dwell period, the buffer can be replaced with freshbuffer. This can be repeated one or more times, for example, a total ofthree buffer baths, before the buffer is replaced with water. As withthe buffer, the water can be introduced to completely fill the body'svascular system and to completely cover the body. After a dwell period,the water can be replaced with fresh water. This may be repeated one ormore times, for example, a total of three water baths.

At 108, the body can be dehydrated by replacing the water with analcohol. When critical point drying 112 is performed using carbondioxide, dehydration 112 is necessary since carbon dioxide is generallyimmiscible in water. The water thus must be replaced with a misciblefluid, e.g., an alcohol, in order for the critical point drying to beeffective. As with the buffer and water, the alcohol can be perfusedthrough and flowed over the body for a sufficient volume to allowcomplete removal of the water. Alternatively, a sufficient volume ofalcohol can be introduced to completely fill the body's vascular systemand to completely cover the body. After a dwell period, the alcohol canbe replaced with fresh alcohol. This may be repeated one or more times,for example, for a total of three alcohol baths.

At 112, liquid carbon dioxide (LCO₂) can be introduced to replace thealcohol in and around the body. The body can be placed in a pressurechamber filled with alcohol, after which the liquid carbon dioxide canbe introduced at a low temperature. After the liquid carbon dioxidereplaces the alcohol in the chamber and the body, the chamber can beheated until the temperature and the pressure in the chamber is at orexceeds the critical point of carbon dioxide, i.e., a temperature of 31°C. and a pressure of 1072 psi. Such temperatures and pressures can beattained without sustaining damage to the cell structure and integrity.

The fixation step 104, flushing step 106, and dehydration step 108 maybe incorporated into a common setup 110. The combinedfixation/flushing/dehydration setup (such as that shown in FIGS. 3-4)can be separate from the critical point drying step 112 such that thecritical point drying step 112 is performed using a different apparatus(such as that shown in FIGS. 5A-5B). The fixation step 104, flushingstep 106, dehydration step 108, and critical drying step 112 can also beincorporated together into a common setup 114, such as that shown inFIG. 6 or FIGS. 7A-7C.

Referring now to FIGS. 2A-2B, an example of a detailed process flow forpreservation of a body is shown. Beginning with FIG. 2A, the process ofblock 200 begins at 202, where an inlet flow path and an outlet flowpath are connected to the vascular system of the body. The flow pathscan include tubing and/or fluid conduits designed to carry fixative andpotentially other fluids, such as buffer, water, alcohol, and liquidcarbon dioxide, to the vascular system of the body. For example, thetubing can have a diameter between 0.25 inches and 0.5 inches.

At 204, the inlet and outlet flow paths can be used to perfuse fixativeinto the vascular system. For example, a fixative can be infused throughthe inlet flow path, while the contents of the vascular system aredrained through the outlet flow path. The fixative can be 1.5%glutaraldehyde in a phosphate buffer or a combination of glutaraldehydeand formaldehyde in a buffer, for example. Perfusion of the fixative canbe accomplished, for example, using low pressure pumping or gravityinduced flow. In applications where preservation of cell structure maybe less important, moderate pressure pumping can be used to reduceinfusion and processing times. At least initially, blood will beconveyed out of the vascular system through the outlet flow path. Theinfusion of fixative continues until the contents of the vascular systemare completely replaced with fixative. The fixative can be maintained inthe vascular system for a predetermined period of time sufficient toresult in fixation of the internal cells of the body. For example, thepredetermined period of time can be one to several days, such as threedays. The precise time may be a function of the size of the body, aswell as other factors, and thus may be subject to variation.

At 206, the body can be immersed in a bath of fixative for apredetermined period of time. For example, the predetermined period oftime can be one to several days, such as three days. Steps 204 and 206can be combined into a single step 208 such that the body is immersed inthe bath of fixative at the same time that fixative flows through thevascular system. In such case, the predetermined period of time for bothsteps 204 and 206 can be the same. It is also noted that steps 204 and206 can be transposed such that step 206 occurs before step 204.

It may be advantageous to renew the fixative within and in contact withthe body. Thus, steps 204 and 206 can be repeated several times (e.g.,three times), each time with a new batch of fixative maintained withinand in contact with the body for a predetermined time period (e.g., oneday). At 210, it is determined if steps 204 and 206 should be repeated.If it is determined that the fixation should be repeated, the processproceeds to 212, where a new batch of fixative is prepared, and steps204 and 206 are then repeated. Otherwise, the process proceeds to 214.

At 214, the inlet and outlet flow paths can be used to perfuse bufferinto the vascular system. For example, a buffer, such as phosphatebuffer, can be infused through the inlet flow path, while the contentsof the vascular system arc drained through the outlet flow path. Atleast initially, fixative will be conveyed out of the vascular systemthrough the outlet flow path. The infusion of buffer continues until thecontents of the vascular system are completely replaced with buffer. Thebuffer can be maintained in the vascular system for a predeterminedperiod of time. For example, the predetermined period of time can bebetween one and two hours. The precise time may be a function of thesize of the body, as well as other factors, and thus may be subject tovariation.

At 216, the body can be immersed in a bath of buffer for a predeterminedperiod of time. For example, the predetermined period of time can be oneto two hours. Steps 214 and 216 can be combined into a single step 218such that the body is immersed in the bath of buffer at the same timethat buffer flows through the vascular system. In such case, thepredetermined period of time for both steps 214 and 216 can be the same.It is also noted that steps 214 and 216 can be transposed such that step216 occurs before step 214.

It may be advantageous to renew the buffer within and in contact withthe body. Thus, steps 214 and 216 can be repeated several times (e.g.,three times), each time with a new batch of buffer maintained within andin contact with the body for a predetermined time period (e.g., 1.5hours). At 220, it is determined if steps 214 and 216 should berepeated. If it is determined that the buffer flush should be repeated,the process proceeds to 222, where a new batch of buffer is prepared,and steps 214 and 216 are then repeated. Otherwise, the process proceedsto 224.

At 224, the inlet and outlet flow paths can be used to perfuse waterinto the vascular system. For example, purified water, such as deionized(DI) water or distilled water, is infused through the inlet flow path,while the contents of the vascular system are drained through the outletflow path. At least initially, buffer will be conveyed out of thevascular system through the outlet flow path. The infusion of watercontinues until the contents of the vascular system are completelyreplaced with water. The water can be maintained in the vascular systemfor a predetermined period of time. For example, the predeterminedperiod of time can be between one and two hours. The precise time may bea function of the size of the body, as well as other factors, and thusmay be subject to variation.

At 226, the body can be immersed in a bath of water for a predeterminedperiod of time. For example, the predetermined period of time may be oneto two hours. Steps 224 and 226 can be combined into a single step 228such that the body is immersed in the bath of water at the same timethat water flows through the vascular system. In such case, thepredetermined period of time for both steps 224 and 226 can be the same.It is also noted that steps 224 and 226 can be transposed such that step226 occurs before step 224.

It may be advantageous to renew the water within and in contact with thebody. Thus, steps 224 and 226 can be repeated several times (e.g., threetimes), each time with a new batch of water maintained within and incontact with the body for a predetermined time period (e.g., 1.5 hours).At 230, it is determined if steps 224 and 226 should be repeated. If itis determined that the water flush should be repeated, the processproceeds to step 232, where a new batch of water is prepared, and steps224 and 226 are then repeated. Otherwise, the process proceeds to theprocess of block 234 in FIGS. 2A-2B.

Referring now to FIG. 2B, the process proceeds from block 200 to 236. At236, the inlet and outlet flow paths can be used to perfuse an alcoholinto the vascular system. For example, an alcohol, such as isopropylalcohol (TPA) or ethanol, can be infused through the inlet flow path,while the contents of the vascular system are drained through the outletflow path. At least initially, water will be conveyed out of thevascular system through the outlet flow path. The infusion of alcoholcontinues until the contents of the vascular system are completelyreplaced with alcohol. The alcohol can be maintained in the vascularsystem for a predetermined period of time. For example, thepredetermined period of time can be between one and several days, suchas one day. The precise time may be a function of the size of the body,as well as other factors, and thus may be subject to variation.

At 238, the body can be immersed in a bath of alcohol for apredetermined period of time. For example, the predetermined period oftime can be between one and several days. Steps 236 and 238 can becombined into a single step 240 such that the body is immersed in thebath of alcohol at the same time that alcohol flows through the vascularsystem. In such case, the predetermined period of time for both steps236 and 238 can be the same. It is also noted that steps 236 and 238 canbe transposed such that step 238 occurs before step 236.

It may be advantageous to renew the alcohol within and in contact withthe body. Thus, steps 236 and 238 can be repeated several times (e.g.,three times), each time with a new batch of alcohol maintained withinand in contact with the body for a predetermined time period (e.g., oneday). At step 242, it is determined if steps 236 and 238 should berepeated. If it is determined that the dehydration with alcohol shouldbe repeated, the process proceeds to step 244, where a new batch ofalcohol is prepared, and steps 224 and 226 are then repeated.

At 246, the body and alcohol can be cooled in a sealed chamber to afirst predetermined temperature, at which a transitional fluid exists asa liquid, in preparation for critical point drying. When steps 204-238are performed using an apparatus separate from a critical point dryingapparatus, the body can be transferred to the chamber in a bath ofalcohol and sealed to prevent reintroduction of water. The transitionalfluid can be, for example, carbon dioxide, in which case the sealedchamber is cooled to a temperature less than or equal to 10° C. Thepressure can be maintained substantially at atmospheric pressure.

At 248, the inlet flow path is used to perfuse transitional fluid intothe vascular system. A common or separate inlet in the sealed chambercan be used to convey transitional fluid into the sealed chamber so asto replace the alcohol in and surrounding the body. For example,transitional fluid, such as liquid carbon dioxide, can be simultaneouslyinfused through the inlet flow path and introduced into the sealedchamber. The contents of the vascular system can be drained through theoutlet flow path. Since the body is located in a sealed chamber, theoutlet flow path can empty directly into the sealed chamber, oralternatively, be connected to an outlet of the sealed chamber forremoval. An outlet in the sealed chamber may serve to remove the fluidcontents of the sealed chamber as fluid is added thereto.

The infusion of liquid carbon dioxide can continue at least until thecontents of the vascular system are completely replaced with liquidcarbon dioxide. The liquid carbon dioxide can be maintained in thesealed chamber and the vascular system for a predetermined period oftime. Alternatively, the liquid carbon dioxide can be continuouslyflowed through the sealed chamber and the vascular system for apredetermined period of time. For example, the predetermined period oftime can be between ten minutes and one hour. The precise time may be afunction of the size of the body, as well as other factors, and thus maybe subject to variation. During the infusion of liquid carbon dioxide,the chamber and the body therein can be maintained at or below 10° C.

At 250, the flow of liquid carbon dioxide through the sealed chamber andvascular system can be stopped such that the sealed chamber and vascularsystem are filled with liquid carbon dioxide. The body and the carbondioxide in the sealed chamber can then be heated to or beyond thecritical point. The critical point for carbon dioxide occurs at atemperature of approximately 31° C. and a pressure of approximately 1072psi. At the critical point, the density of the liquid and gas phases areidentical such that the carbon dioxide can be exhausted without damageto the cell structure of the body.

At 252, the pressurized carbon dioxide in the sealed chamber can beremoved. Because of the relatively high pressure, the carbon dioxide canbe slowly vented from the sealed chamber while maintaining thetemperature above 31° C. to avoid condensation of the carbon dioxide.For example, the chamber can be vented such that the carbon dioxide isexhausted at a rate of between 8 standard ft³ per hour (SCFH) and 10SCFH. At pressures below 400 psi, for example, the exhaust rate can beincreased, if so desired. When the pressure in the sealed chamber issubstantially equal to atmospheric pressure, the process proceeds to254, wherein the preserved body can be removed from the sealed chamber.

In one or more embodiments, the fixation, flushing, and dehydrationcomponents of the above noted process can be performed using a singleapparatus while the critical point drying is performed using a separateapparatus. For example, FIG. 3 shows an embodiment of a gravityperfusion setup 300 that can be used to perform the fixation, flushing,and dehydration components of the preservation process. Body 302 can beimmersed in a bath 304. As described above, bath 304 can be filled withfixative, buffer, water, or alcohol, depending on the specific processstep. An incision 312 can be made in the thoracic cavity of body 302 toprovide access to the body's heart 314. A syringe or other fluidcontainer 306 (such as an intravenous bag), which is filled with thefluid to be perfused, can be connected via infusion line 308 to thebody's heart. The fluid in bath 304 can be the same as the fluid incontainer 306, i.e., fixative, buffer, water, or alcohol. Gravity causesthe fluid in the container 306 to flow into the vascular system of thebody via the heart, thereby displacing fluid currently in the vascularsystem from the body. Similarly, a drainage line 310 for removing fluidfrom the vascular system is connected to the body's heart. The infusionline 308 can have a needle or cannula, which serves as inlet flow path,connected to the left ventricle of the heart. Drainage line 310 can alsohave a needle or cannula, which serves as an outlet flow path, connectedto the right ventricle of the heart.

In an alternative, the perfusion flow paths can be coupled to thevascular system through one or more blood-line accesses in the body'ssurface. For example, perfusion can be achieved by injection through theright common carotid artery with fluid being drained through the rightjugular vein. For cases where damage or clotting has compromised theability of the vascular system to adequately distribute fluid throughoutthe body, multiple access sites can be used. For example, multipleinjections can be made through the two iliac or femoral arteries,subclavian or axillary vessels, and common carotids. Of course, otheraccess points for introducing fluid into and removing fluid from thevascular system of the body are also possible according to one or morecontemplated embodiments.

While gravity perfusion provides a low-cost option for introducingfluids into the vascular system of a body, perfusion systems withmanually or automatically controllable pumps may provide better and morerepeatable control of perfusion. In addition, multiple perfusion fluids(e.g., fixative, buffer, water, and alcohol) can be integrated into asingle setup to allow seamless and automatic transition between thepreservation steps. A schematic of such a perfusion system 400 is shownin FIG. 4.

Perfusion system 400 includes a fluid source 408, which can includeseparate sources for each of the fluids used in preservation of thebody. Alternatively, the fluid source 408 can be a single source offluid, which is replaced for each phase of the preservation process. Apump 410 controls infusion of the fluid source into a body 402. Fluidfrom the fluid source 408 can be provided to the vascular system 406 ofthe body 402. In addition, the pump 410 can provide the fluid from thefluid source to the external surfaces 404 of the body 402. Externalsurfaces 404 of the body 402 can include the epithelial cells of theintegumentary system (such as the external facing skins cells) as wellas the epithelial cells of the gastrointestinal tract.

The pump 410 can be connected to an inlet of a bath in which the body402 is submerged. A pump 412 can also be provided to assist in theremoval of fluid from the body 402 and/or bath. For example, pump 412can remove fluid from the vascular system 406 to waste 414. Pump 412 canalso be connected to an outlet of a bath in which the body 402. Pumps410 and 412, acting separately or together, can thus continuouslyexchange fluid within and in contact with the body with fresh or newfluid from the fluid source. Pumps 410 and 412 can be, for example, aninfusion pump, a peristaltic pump, a syringe pump, or any other knownpump. Of course, pump 410 or pump 412 may be omitted and/or other pumpsmay be added to achieve similar fluid flow configurations according toone or more contemplated embodiments.

Referring now to FIGS. 5A-5B, an embodiment of a pressure chamber 500for critical point drying of a body is shown. Pressure chamber 500 isshown in plan (i.e., top down) view in FIG. 5A and in cross-sectionalong line B-B in FIG. 5B. Pressure chamber 500 includes multiplesidewalls 506, a bottom wall 530, and a top access panel 534. Accesspanel 534 (not show in FIG. 5A) can include a viewing window 536 so thatan operator can monitor the critical point drying process. An O-ring 538can extend around the perimeter of the access panel 534 so as to providea seal. Various mechanisms (not shown) can be employed to secure thepanel 534 to the side walls 506 of the pressure chamber 500.

The pressure chamber 500 can include at least two fluid ports 508, whichcan serve as an inlet or an outlet. Fluid ports 508 can be provided inone or more of the sidewalls 506. Alternatively or additionally, thepressure chamber 500 can include one or more fluid ports 510, which canserve as an inlet or an outlet. Fluid ports 510 can be provided in thebottom wall 530. The side walls 506 together with the bottom wall 530and the top panel 534 define an internal volume 504, in which the body502 is placed. Fluid (e.g., carbon dioxide) can be introduced into thevolume 504 through one or more fluid ports 508/510 acting as inlets andcan be removed from the volume 504 through one or more fluid ports508/510 acting as outlets. The volume 504 is sized and shaped toaccommodate the body to be preserved plus a certain volume of fluid(e.g., carbon dioxide). When the body 502 is a human body, the internalvolume 504 may be between 100 L and 600 L. With such a large volume,multiple ports 508/510 can be provided in the respective sidewalls 506and bottom wall 530 to allow efficient introduction and removal ofcarbon dioxide and other fluids.

An inlet connection 518 can also be provided in the interior volume forconnecting an infusion line 520 (i.e., vascular inlet line) forintroducing fluid (e.g., carbon dioxide) into the vascular system of thebody 502. The infusion line 520 may be connected to the heart 522 of thebody, for example, through the left ventricle. Similarly, an optionaloutlet connection 524 can also be provided in the interior volume forconnecting a discharge line 528 for removing fluid from the vascularsystem. The discharge line 528 can be connected to the heart 522 of thebody, for example, through the right ventricle. However, the dischargeline 528 need not be connected to a separate outlet 524, but candischarge directly into the interior volume 504. Alternatively, thedischarge line can be omitted in favor of a direct discharge from thevascular system into the interior volume. As explained above, theconnection to the vascular system is not required to be through theheart. Rather, other connections, such as through arterial and venousaccess points on the exterior of the body, are also possible. Where morethan one access point is used, more than one infusion line 520 withrespective inlet 518 or more than one discharge line 528 with respectiveoutlet 524 can be provided in the pressure chamber 500.

Pressure chamber 500 can also include one or more additional fluid linesfor infusing fluid (e.g., carbon dioxide) through generally inaccessibleportions of the body. For example, when connection to the vascularsystem is provided through the heart, the incision in the thoraciccavity can be left open such that fluid exchange between the thoracicand peritoneal cavities and the internal volume of the pressure chamberis possible. However, when connection to the vascular system is providedthrough arterial or venous accesses, separate fluid lines may benecessary to allow fluid (e.g., carbon dioxide) introduction to andremoval from the thoracic and/or peritoneal cavities. In addition,passive fluid exchange between the gastrointestinal tract and theinternal volume of the pressure chamber may be insufficient.Accordingly, one or more fluid lines can be coupled to thegastrointestinal tract to flow fluid (e.g., carbon dioxide)therethrough. For example, a fluid line 514 can be connected to an inletconnection 512 in the interior volume 504 to provide fluid (e.g., carbondioxide) to the upper gastrointestinal tract through the mouth 516 ofthe body 502. A separate line (not shown) can provide fluid (e.g.,carbon dioxide) to the lower gastrointestinal tract through the anus ofthe body 502. Intermittent introduction of fluid (e.g., carbon dioxide)coupled with intermittent removal, or continuous introduction andremoval of fluid (e.g., carbon dioxide) through a single connection, canbe achieved using a setup similar to that employed in colonhydrotherapy.

It is desirable to circulate fluid (e.g., carbon dioxide) to allportions of the body to allow for appropriate surface treatment andremoval of all previous fluids (e.g., alcohol). A support 532 can beprovided to raise the body 502 off of the bottom wall 530 and to anintermediate height within the interior volume 504. The support 532 caninclude holes, openings, dimples, or flow channels, or be substantiallyporous so as to allow fluid to reach substantially all points on theexterior surface of the body 502. For example, the support 532 can be aporous wire mesh. Other supports that allow the fluids to reachsubstantially all surfaces of the body 502 are also possible accordingto one or more contemplated embodiments.

It should be noted that the pressure chamber illustrated in FIGS. 5A-5Bis not restricted to use in the critical point drying component of thepreservation process. Rather, it may be used to perform the fixation,flushing, and dehydration components in addition to the critical pointdrying component of the preservation process, as discussed with respectto FIGS. 6 and 7A-7C below.

Referring now to FIG. 6, a schematic diagram of an automated system 600for preservation of a body is shown. The automated system 600 caninclude a pressure chamber 602, a control system 604, and a fluid supplysystem 606. Pressure chamber 602 can be substantially similar to thatpresented in FIGS. 5A-5B and discussed in detail above. In general, thepressure chamber 602 can be sized and shaped to accommodate a desiredbody size or range of body sizes.

Fluid supply system 606 can include one or more fluid containers withappropriate solutions for performing the preservation process describedherein. For example, the fluid supply system 606 can include supplies offixative 628, buffer 630, water 632, alcohol 634, and/or liquid carbondioxide 636. Appropriate fluidic connections 638 can couple the fluidsupply system, and the individual supplies therein, to the fluid controlmodule 608 of the control system 604.

Control system 604 can include multiple control modules. For example,control system 604 can include a fluid control module 608, a centralcontrol module 610, and/or a temperature control module 612. Althoughshown as separate modules 608-612, the control modules of the controlsystem 604 can be embodied in a single unit, such as a specializedcomputer or processor. In addition, although the control modules 608-612are illustrated as part of a single control system 604, they can beembodied as completely separate and individual units. Each module608-612 can be a stand-alone computer or processor or integrated withother components. For example, temperature control module 612 can beintegrated with a heating and cooling system that provides the necessarytemperature adjustment of the pressure chamber 602. In another example,fluid control module 608 can be integrated with a fluid pump or valvesystem that provides control over the fluid delivered to the pressurechamber 602. In such configurations, the central control module 610 cancommunicate with fluid control module 608 and temperature control module612 through wired or wireless connections to provide control of thepressure chamber 602 environment during the preservation process.

The fluid control module 608 can include pumps and/or valves to controlthe flow of fluid from the fluid source 606 to the pressure chamber 602.For example, the fluid control module 608 can select the appropriatefluid supply 628-636 in the fluid supply 606 and, through appropriatecontrol of any valves and pumps, can convey the selected fluid alonginput line 614 to the pressure chamber. Fluid control module 608 canalso monitor the waste line 626 and/or the vent line 624 of the pressurechamber 602 in order to control flow therethrough. For example, thefluid control module 608 can regulate the flow of gaseous carbon dioxideexhaust through the vent line 624 to prevent undesirable condensation inthe pressure chamber 602 after the heating step.

Temperature control module 612 can include appropriate heating orcooling elements, such as resistive heaters, thermoelectric coolers,refrigeration elements, and temperature sensors, such as thermocouples,thermoresistors, or infrared detectors, to control the temperature ofthe fluid in the pressure chamber 602. Alternatively, appropriateheating/cooling elements and sensors can be integrated with the pressurechamber 602 and communicate with the temperature control module throughdata line 622. For example, the temperature control module 612 can coolthe pressure chamber 602 prior to and during the introduction of liquidcarbon dioxide. The liquid carbon dioxide can be used to assist incooling the pressure chamber. In addition, the temperature controlmodule 612 can heat the pressure chamber 602 with the liquid carbondioxide therein to the critical point. The temperature control module612 can also control the temperature in the pressure chamber duringother aspects of the preservation process, such as cooling the pressurechamber during the fixation procedure when the progression of decay canbe inhibited by the reduced temperature.

Central control module 610 can regulate operation of the fluid controlmodule 608 through data line 616 and the temperature control module 612through data line 620 to perform the preservation process describedabove with respect to FIGS. 2A-2B. Control module 610 can alsocommunicate with the pressure chamber 602 via data line 602, forexample, to monitor the condition of the pressure chamber. For example,the control module 610 can halt or interrupt the preservation process ifa sensor on the pressure chamber 602 indicates that an access panel hasbeen opened or that a connection to the vascular system has beendisturbed. Central control module 610 can also respond to inputs from anoperator, for example, through a visual touch-screen display, displayand keyboard, or other conventional input/output devices.

Referring now to FIGS. 7A-7C, embodiments of an integrated preservationsystem are shown. In FIG. 7A, a preservation system 700A can include afluid cabinet 706 to supply fluid to a pressure chamber 702A. Controller704 can control fluid flow to the pressure chamber 702A through fluidinput line 708. Access panel 714A of the pressure chamber can have awindow 710A for observing the body 712 during the fixation, flushing,dehydration, and critical point drying phases of the preservationprocess. The pressure chamber 702A can be constructed such that the body712 is maintained upright throughout the process. Access panel 714A canbe hinged and opened like a door to allow removal of the body 712 fromthe pressure chamber 702A.

FIG. 7B illustrates an alternative preservation system 700B, which issimilar to that of FIG. 7A. However, pressure chamber 702B has ahorizontal configuration as opposed to the vertical configuration ofchamber 702A. Body 712 can be positioned supine on a movable support,similar to storage systems in a conventional mortuary. Window 710B inaccess panel 714B can allow for observation of the body 712 during thefixation, flushing, dehydration, and critical point drying phases of thepreservation process.

FIG. 7C illustrates yet another alternative preservation system 700C,which is similar to that of FIG. 7B. In further contrast to chamber702B, pressure chamber 702C has the opening to the interior volume atthe top thereof. Access panel 714C can rotate about a horizontal axisfrom a substantially horizontal position to a substantially verticalposition to allow access to the interior volume and a body 712 therein.Although not shown, access panel 714C can include a window for viewingthe body 712 during the fixation, flushing, dehydration, and criticalpoint drying phases of the preservation process.

The embodiments of FIG. 7A-7C illustrate exemplary configurations for anintegrated preservation system, but configurations for an integratedpreservation system are not intended to be limited to these illustratedembodiments. Rather, other configurations and arrangements for anintegrated preservation system are contemplated in accordance with theteachings of the present disclosure.

As discussed above, fixation is not necessary to effect preservation ofall tissue structures. Thus, the process of FIGS. 2A-2B can be modifiedto remove fixation steps 204-210. Alternatively, the process can bemodified to perform critical point drying alone with only a dehydrationstep. Referring now to FIG. 8, an alternative preservation process 800is shown. At 802, an inlet flow path and an outlet flow path can beconnected to the vascular system of the body. The flow paths can includetubing and/or fluid conduits designed to carry fixative and potentiallyother fluids, such as buffer, water, alcohol, and liquid carbon dioxide,to the vascular system of the body. For bodies or tissues that do nothave a vascular system, such as vegetation or non-vascular organisms,step 802 and the following perfusion steps may not be necessary.

At 804, the inlet and outlet flow paths can be used to perfuse analcohol into the vascular system. For example, an alcohol, such asisopropyl alcohol (IPA) or ethanol, can be infused through the inletflow path, while the contents of the vascular system can be drainedthrough the outlet flow path. At least initially, blood and/or othervascular fluid will be conveyed out of the vascular system through theoutlet flow path. The infusion of alcohol can continue until thecontents of the vascular system are completely replaced with alcohol.The alcohol can be maintained in the vascular system for a predeterminedperiod of time. For example, the predetermined period of time can bebetween one and several days, such as one day. The precise time may be afunction of the size of the body, as well as other factors, and thus maybe subject to variation.

At 806, the body can be immersed in a bath of alcohol for apredetermined period of time. For example, the predetermined period oftime can be between one and several days. Steps 804 and 806 can becombined into a single step 808 such that the body is immersed in thebath of alcohol at the same time that alcohol flows through the vascularsystem. In such case, the predetermined period of time for both steps804 and 806 can be the same. It is also noted that steps 804 and 806 canbe transposed such that step 806 occurs before step 804.

It may be advantageous to renew the alcohol within and in contact withthe body. Thus, steps 804 and 806 can be repeated several times (e.g.,three times), each time with a new batch of alcohol maintained withinand in contact with the body for a predetermined time (e.g., one day).At step 810, it is determined if steps 804 and 806 should be repeated.If so, the process proceeds to step 812, where a new batch of alcohol isprepared, and steps 804 and 806 are repeated. Otherwise, the processproceeds to 814.

At 814, the body and alcohol can be cooled in a sealed chamber to afirst predetermined temperature at which a transitional fluid exists asa liquid in preparation for critical point drying. When steps 804-810are performed using an apparatus separate from a critical point dryingapparatus, the body can be transferred to the chamber in a bath ofalcohol and sealed to prevent reintroduction of water. The transitionalfluid can be, for example, carbon dioxide, in which case the chamber canbe cooled to a temperature less than or equal to 10° C.

At 816, the inlet flow path can be used to perfuse transitional fluidinto the vascular system. A common or separate inlet in the sealedchamber can be used to convey transitional fluid into the sealed chamberso as to replace the alcohol and surround the body. For example,transitional fluid, such as liquid carbon dioxide, can be infusedthrough the inlet flow path and into the sealed chamber. The contents ofthe vascular system can be simultaneously drained through the outletflow path. Since the body is located in a sealed chamber the outlet flowpath can empty directly into the sealed chamber or be connected to anoutlet of the sealed chamber. An outlet in the sealed chamber can serveto remove the fluid contents of the sealed chamber as fluid is addedthereto.

The infusion of liquid carbon dioxide continues at least until thecontents of the vascular system are completely replaced with liquidcarbon dioxide. The liquid carbon dioxide can be maintained in thesealed chamber and the vascular system for a predetermined period oftime. Alternatively, the liquid carbon dioxide can be continuouslyflowed through the sealed chamber and the vascular system for apredetermined period of time. For example, the predetermined period oftime can be between ten minutes and one hour. The precise time may be afunction of the size of the body, as well as other factors, and thus maybe subject to variation.

At 818, the flow of liquid carbon dioxide through the sealed chamber andvascular system can be stopped such that the sealed chamber and vascularsystem are filled with liquid carbon dioxide. The body and the carbondioxide in the sealed chamber can then be heated to or beyond thecritical point. At 820, the pressurized carbon dioxide in the sealedchamber can be removed. Because of the relatively high pressure, thecarbon dioxide can be slowly vented from the sealed chamber whilemaintaining the temperature above 31° C. to avoid condensation of thecarbon dioxide. For example, the chamber can be vented such that thecarbon dioxide is exhausted at a rate of between 8 SCFH and 10 SCFH. Atpressures below 400 psi, for example, the exhaust rate can be increased,if desired. When the pressure in the sealed chamber is substantiallyequal to atmospheric pressure, the process proceeds to 822, wherein thepreserved body can be removed from the sealed chamber.

As decomposition begins immediately upon death, it may be preferablethat the methods disclosed herein be applied as soon after death aspossible. For example, a recently-deceased body may be perfused within15 minutes of death. Such a time period may be necessary when it isdesired to preserve cell structure for clinical or medical examination,research, or reanimation preservation. For purely funereal and cosmeticapplications where it may not be necessary to maintain all cellstructure or integrity, a recently-deceased body may be perfused within5 hours of death. Of course, these time figures are exemplary andvariations therefrom are contemplated. For example, the maximum timebefore perfusion may be affected by the environment in which the body ismaintained, as heat, humidity, and exposure to bacteria or scavengersmay accelerate decomposition.

Although particular configurations have been discussed herein, otherconfigurations can also be employed. Moreover, although specificchemicals have been discussed, other chemicals may be used to achievethe desired effect. For example, other fixatives, such as, but notlimited to, formaldehyde, paraformaldehyde, osmium tetroxide, uranylacetate, acrolein, glutaraldehyde, and combinations thereof as well aspurified versions thereof are possible according to contemplatedembodiments of the disclosed subject matter. In another example, othertransitional fluids, such as, but not limited to, hydrogen, oxygen,nitrogen, and carbon monoxide, are also possible according tocontemplated embodiments of the disclosed subject matter.

All chemicals and compositions described herein are for illustrationpurposes only and should not be understood as limiting of theembodiments of the disclosed subject matter. Furthermore, the foregoingdescriptions apply, in some cases, to examples generated in alaboratory, but these examples can be extended to production techniques.For example, where quantities and techniques apply to the laboratoryexamples, they should not be understood as limiting.

It is, thus, apparent that there is provided, in accordance with thepresent disclosure, critical point drying systems and methods for insitu tissue preservation. Many alternatives, modifications, andvariations are enabled by the present disclosure. Features of thedisclosed embodiments can be combined, rearranged, omitted, etc., withinthe scope of the invention to produce additional embodiments.Furthermore, certain features may sometimes be used to advantage withouta corresponding use of other features. Accordingly, Applicant intends toembrace all such alternatives, modifications, equivalents, andvariations that are within the spirit and scope of the presentinvention.

1-23. (canceled)
 24. A system for preservation of a body or an organthereof, the system comprising: a pressure chamber having at least onefluid inlet and a vascular inlet line, the vascular inlet line beingconstructed to connect to the vascular system of the body or the organ,the pressure chamber being sized and shaped so as to allow the body orthe organ to be enclosed therein; a flow module configured to supplyliquid carbon dioxide to the pressure chamber through the at least onefluid inlet and to the vascular system through the vascular inlet line;a temperature module configured to control the temperature of thepressure chamber; and a system controller configured to control the flowmodule to fill the pressure chamber and the vascular system with liquidcarbon dioxide and to control the temperature module to heat the liquidcarbon dioxide in the chamber and the vascular system above the carbondioxide critical point temperature and pressure, wherein the pressurechamber is constructed to withstand a temperature and pressure of atleast said carbon dioxide critical point temperature and pressure. 25.The system of claim 24, wherein the vascular inlet line is constructedto connect to the vascular system of the body by way of one of theventricles of the heart.
 26. The system of claim 24, wherein the systemcontroller controls the temperature module to cool the pressure chamberprior to the chamber being filled with liquid carbon dioxide.
 27. Thesystem of claim 24, wherein the flow module is further configured tosupply a fixative to the pressure chamber through the at least one fluidinlet and to the vascular system through the vascular inlet line, andthe system controller controls the flow module to fill the chamber andthe vascular system with fixative prior to filling the chamber and thevascular system with liquid carbon dioxide.
 28. The system of claim 24,wherein the fixative includes at least one of glutaraldehyde andformaldehyde.
 29. The system of claim 24, wherein the pressure chamberhas a volume between 100 L and 600 L.
 30. The system of claim 24,wherein the body or the organ is of an animal.
 31. The system of claim30, wherein the body or the organ is of a human.
 32. A method forpreserving a body or an organ thereof, the method comprising: providinga preservation system, the preservation system comprising: a pressurechamber having at least one fluid inlet and a vascular inlet line, thepressure chamber being sized and shaped so as to allow the body or theorgan to be enclosed therein; a flow module configured to supply atleast liquid carbon dioxide via the fluid inlet and the vascular inletline; a temperature module configured to control the temperature of thepressure chamber; and a system controller configured to control the flowmodule and the temperature module, the pressure chamber beingconstructed to withstand a temperature and a pressure of at least acritical point temperature and pressure for carbon dioxide; perfusingthe body or the organ with liquid carbon dioxide by way of the vascularinlet line connected to the vascular system of the body or organ whilethe body or the organ is in the pressure chamber; exposing externalsurfaces of the body or the organ to liquid carbon dioxide supplied viasaid at least one fluid inlet while the body or the organ is in thepressure chamber; heating the body or the organ in the sealed pressurechamber until the temperature and pressure in said pressure chamber meetor exceed the critical point for carbon dioxide; and after the heating,exhausting carbon dioxide from the pressure chamber and the body or theorgan therein.
 33. The method of claim 32, wherein the exposing toliquid carbon dioxide is performed simultaneously with the perfusingliquid carbon dioxide.
 34. The method of claim 33, wherein the exposingto liquid carbon dioxide includes flowing liquid carbon dioxide over theexternal surfaces of the body or the organ.
 35. The method of claim 32,wherein the external surfaces include the epithelial cells of theintegumentary system and the gastrointestinal tract of the body.
 36. Themethod of claim 32, further comprising: prior to the perfusing withliquid carbon dioxide, perfusing the body or the organ with fixative byway of the vascular inlet line connected to the vascular system of thebody or organ; and at a same time as the perfusing with fixative,exposing external surfaces of the body or organ to fixative supplied viasaid at least one fluid inlet while the body or organ is in the pressurechamber.
 37. The method of claim 32, wherein the body or the organ is ofan animal.
 38. The method of claim 37, wherein the body or the organ isof a human.
 39. A method for preserving a recently deceased human body,the method comprising: (a) providing a preservation system, thepreservation system comprising: a pressure chamber having at least onefluid inlet and a vascular inlet line, the pressure chamber being sizedand shaped so as to allow the deceased human body to be enclosedtherein; a flow module configured to supply at least liquid carbondioxide and a fixative via the fluid inlet and the vascular inlet line;a temperature module configured to control the temperature of thepressure chamber; and a system controller configured to control the flowmodule and the temperature module, the pressure chamber beingconstructed to withstand a temperature and a pressure of at least acritical point temperature and pressure for carbon dioxide; (b)connecting the vascular inlet line to the vascular system of thedeceased human body; (c) after (b), flowing the fixative into thevascular system of the deceased human body and into the pressure chambervia the fluid inlet so as to immerse the deceased human body infixative, the fixative including at least one of glutaraldehyde andformaldehyde; (d) after (c), flowing a buffer solution into the vascularsystem and into the pressure chamber via the fluid inlet so as toimmerse the deceased human body in buffer solution; (e) after (d),flowing water into the vascular system and into the pressure chamber viathe fluid inlet so as to immerse the deceased human body in water; (f)after (e), flowing alcohol into the vascular system and into thepressure chamber via the fluid inlet so as to immerse the deceased humanbody in alcohol; (g) after (f), flowing liquid carbon dioxide into thevascular system and into the pressure chamber via the fluid inlet so asto immerse the deceased human body in liquid carbon dioxide; and (h)after (g), heating the deceased human body in the pressure chamber untilthe temperature and pressure in said pressure chamber meet or exceed thecritical temperature and critical pressure for carbon dioxide.
 40. Themethod of claim 39, wherein the connecting includes opening the deceasedhuman body's thoracic cavity so as to access the heart, wherein thecavity remains open between the connecting and the heating.
 41. Themethod of claim 39, wherein the connecting includes connecting thevascular inlet line to the left ventricle of the heart and a vascularoutlet line to the right ventricle of the heart.
 42. The method of claim39, wherein the connecting includes connecting the vascular inlet lineto an arterial port or access and a vascular outlet line to a venousport or access.
 43. The method of claim 39, wherein (c) through (h)occur with the human body sealed within the pressure chamber.
 44. Themethod of claim 39, wherein the fixative includes 1.5% glutaraldehyde ina buffer, and the alcohol includes isopropyl alcohol or ethanol.